More than 65 years ago Mandel and Metais described for the first time their observation of the presence of extracellular nucleic acids in humans (Mandel P, Metais P. Les acides nucleiques du plasma sanguin chez l'homme. C. R. Acad. Sci. Paris 142, 241-243. 1948) and more than four decades later it could be clearly demonstrated that tumor-associated genetic alterations can be found in cell-free nucleic acids isolated from plasma, serum and other body fluids (Fleischhacker M, Schmidt B. (2007) Circulating nucleic acids (CNAs) and cancer—a survey. Biochim Biophys Acta 1775: 181-232; Jung K, Fleischhacker M, Rabien A. (2010) Cell-free DNA in the blood as a solid tumor biomarker—a critical appraisal of the literature. Clin Chim Acta 411: 1611-1624). This includes epigenetic alterations observed in different forms of malignant tumors. A hallmark of mammalian chromatin is DNA methylation and it is known that cytosine methylation in the context of a CpG dinucleotide plays a role in the regulation of development and is important in basic biological processes like embryogenesis and cell differentiation (Smith Z D, Meissner A. (2013) DNA methylation: roles in mammalian development. Nat Rev Genet 14: 204-220; Gibney E R, Nolan C M. (2010) Epigenetics and gene expression. Heredity (Edinb) 105: 4-13). As such, methylation not only regulates gene transcription, but also plays a role in maintaining genome stability, imprinting and X-chromosome inactivation. Epigenetic alterations in oncogenes and tumor suppressor genes are of key importance in the development of cancer (Suva M L, Riggi N, Bernstein B E. (2013) Epigenetic reprogramming in cancer. Science 339: 1567-1570). DNA methylation patterns are largely modified in cancer cells and can therefore be used to distinguish cancer cells from normal tissues. As such, DNA methylation patterns are being used to diagnose all sorts of cancers. A relatively recent concept is the use of free circulating tumor DNA that is released from the tumor for example into the blood for methylation analysis as an indicator for tumor load in the body of the patient. This ability to isolate and to characterize extracellular nucleic acids from tumor patients with very sensitive and highly specific methods led to the term “liquid biopsy”. As a result, physicians no longer depend exclusively on a single examination of tissue biopsies and body scans. The detection of small amounts of methylated tumor DNA with high backgrounds of unmethylated non-tumor DNA in such a liquid biopsy greatly challenges the sensitivity of the detection methods. Very good results have been achieved using technologies based on a selective amplification of methylated tumor DNA after bisulfite conversion, like methylation-specific PCR (MSP) and especially the HeavyMethyl™ (HM) technology (Cottrell et al., A real-time PCR assay for DNA-methylation using methylation-specific blockers. Nucleic Acids Res. 2004 Jan. 13; 32(1):e10).
The less advanced the cancer is, the better the treatment options and the chances of curing the patient are. Thus, it is highly desirable to diagnose a cancer as early as possible. However, a less advanced cancer, which means smaller tumor size and less cancer cells, releases less free circulating tumor DNA. This is exacerbated by the fact that the half-life of extracellular nucleic acids is rather short, for example less than six hours in plasma (Rago C, Huso D L, Diehl F, Karim B, Liu G, et al. (2007) Serial assessment of human tumor burdens in mice by the analysis of circulating DNA. Cancer Res 67: 9364-9370). Therefore, the more sensitive a detection method is, the earlier the cancer can be reliably diagnosed or diagnosed at all. Accordingly, there is a need in the art for methods of detecting DNA methylation with an increased sensitivity.
For sensitive detection, HeavyMethyl™ or HM (primers bind methylation unspecifically but are blocked methylation specifically and therefore will be re-blocked even if unwanted template is produced at minimal levels when blocking failed in previous amplification cycles) is regarded as first choice when compared to MSP (which primes methylation specifically and therefore introduces perfect match template in case that mispriming happens which will then exponentially be amplified). The advantages of HM seemed to be a tradeoff with one disadvantage: The widely accepted theory was that CpG rich parts for the blockers need to be side by side with priming sites without CpGs. This greatly limits the choice of the site or region to be analysed, because there are only so many suitable sites or regions in a given target DNA.
Due to design constrictions (undesired CpG sites, SNPs etc.), methylation assays in the past used amplicons with sizes up to 150 bp for HM, which the inventors thought to be generally applicable also to address the fragmentation of DNA in circulating tumor DNA. Years of experience and comparison of results showed the inventors that cancer marker assays with such length were very useful in specimen containing cancer cells (full size high molecular weight genomic tumor DNA), and also useful (although with a somewhat reduced sensitivity) when applied to liquid biopsies/body fluids, in which the target is free circulating DNA (such DNA is expected to be at least partially fragmented).
The inventors have now found that, surprisingly, a further decrease in the size of the amplificate leads to a much better amplification (even independent of DNA fragmentation as found in circulating tumor DNA), giving a significantly improved signal which is especially useful when the original template, i.e. the methylated tumor DNA, is scarce among a high background of unmethylated non-tumor DNA. The reduction of the amplicon size as a primary design goal overruling other concerns (such as undesired CpG sites, SNPs etc.) has, to the inventors knowledge, not been done before since a small size greatly limits the choice of sites or regions to be analysed: there are much fewer suitable sites or regions because of the presence of CpG sites which would be covered by the primers, which must be methylation-unspecific for a sensitive detection using HeavyMethyl™. Also, SNP sites should not be covered by the primers, since it biases sensitivity if the primers are specific for a particular nucleotide of the SNP.
The inventors also found, surprisingly, that the introduction of mismatches in CpG sites (or SNP sites) in the primers for HM (methylation unspecific by introducing e.g. C=C, T=C, C=T or T=T mismatches with the first base in the primer and the second in the bisulfite DNA sense template—or A=G, G=G, A=A or G=A mismatches—with the first base in the primer and the second in the bisulfite synthetized reverse complement strand—for cytosine positions in CpG sites) did not introduce worse blocking or unspecific priming even if a mismatch position was located in the middle of the primer (and not limited to positions next to the 5′ end of a primer were one would expect little negative influence), not only when the overall primer binding enthalpy was adjusted by design (e.g. extension). In fact, there was evidence that such constructs might even be blocked better. The inventors believe that this might be due to the instability of blocked primers being even higher compared to unblocked primers still being well annealed.
Methods for detecting cancer which are adapted according to these findings will allow for an improved care for cancer patients by providing the possibility of the most promising time window for treatment.